Thesis for the degree of Doctor of Medicine

Muscarinic Receptors in the Urinary Bladder

The role of the urothelium regarding cholinergic

and nitrergic effects in inflammation

Michael Andersson

2010

Department of Pharmacology

Institute of Neuroscience and Physiology

The Sahlgrenska Academy at the University of Gothenburg

Sweden

Printed by Intellecta Infolog AB, Gothenburg, Sweden, 2010

Previously published papers were reproduced with permission

from the publishers

ISBN 978-91-628-8198-6

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Abstract

Inflammation alters the functional properties of the urinary bladder.

Interstitial cystitis (IC) is a chronic inflammatory syndrome in man

that is characterized by urgency, frequency and visceral pain. The

overall aim of this study was to investigate how the rat urinary

bladder is affected by inflammation, and what specific part the

urothelium plays in this.

Methods: Cystitis was induced in rats by a single injection of

cyclophosphamide (CYP; 100 mg/kg). This treatment causes a

disease state which is highly comparable to IC. Data comparing the

properties of the healthy and inflamed bladder were gathered from

(1) contraction experiments in vitro in an organ bath setup, (2)

cystometrical studies in vivo in anaesthetized rats and (3) wake,

freely moving rats in a metabolism cage. Cell cultures were also

cultivated in order to investigate if proliferation of urothelial cells is

influenced by receptor activation.

Key findings: Induction of cystitis by CYP altered the cholinergic

response of the urinary bladder. In vitro studies showed a

significantly lower response to carbachol in the inflamed bladder.

Both in vitro and in vivo, the altered cholinergic response could be

normalized by either removal of the urothelium, blockade of nitric

oxide (NO) synthase or blockade of muscarinic M1/M3/M5

receptors. These findings indicate that during CYP-induced cystitis,

NO is released from the urothelium upon muscarinic receptor

activation. Further characterization in vitro revealed the M5 receptor

as the most likely candidate for mediating this release.

In vivo experiments in the metabolism cage showed that micturition

parameters are affected by CYP-induced cystitis. Increasing doses of

a muscarinic antagonist eliminated these differences, and a

connection between the effects of antimuscarinic and antinitrergic

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drugs was indicated. These findings underline the importance of

muscarinic receptors and NO in the alterations seen during cystitis.

Proliferation experiments indicated that adrenergic, but not

muscarinic, nicotinic or EGF receptors, are involved in the regulation

of urothelial cell proliferation.

Conclusions: In CYP-induced cystitis in the rat, the urothelium

exerts an inhibitory influence on the cholinergic response of the

urinary bladder. We conclude that this is caused by the release of NO

upon activation of urothelial muscarinic M5 receptors.

Keywords: urinary bladder, inflammation, cyclophosphamide-induced

cystitis, muscarinic receptor, urothelium, nitric oxide, M5 receptor, rat,

proliferation, micturition

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Populärvetenskaplig sammanfattning

Urinblåsan utgör en central del av urinvägarna. Dess uppgifter består

av lagring och tömning av urin. Så länge urinblåsan fungerar som den

ska är man omedveten om dess existens, men när den krånglar kan

man inte låta bli att tänka på den. I Sverige lider runt 400.000

personer av inkontinens, och 4,5% av all diagnostiserad cancer har

sitt ursprung i urinblåsan. Flera av de behandlingar som finns idag är

otillräckliga, vilket visar på behovet av mer kunskap om urinblåsan

och dess sjukdomar. I denna avhandling presenteras ny kunskap om

de förändringar som sker i urinblåsan vid inflammation.

Att urinblåsan blir inflammerad är mycket vanligt och kan bero på

flera orsaker. Till exempel kan inflammation i urinblåsan, s.k. cystit,

orsakas av en bakterieinfektion eller närvaro av kemiska ämnen i

urinen som irriterar urinblåsans insida till den grad att en

inflammation uppstår. Urinblåsans insida täcks av en typ av celler

som kallas urotelceller. Dessa trodde man ända fram till för 10-15 år

sedan var passiva celler med ett enda syfte: att skydda urinblåsan

och resten av kroppen från toxiska ämnen i urinen. Senare forskning

har dock visat att dessa celler har flera viktiga uppgifter, inte minst

vid diverse sjukdomstillstånd. Interstitiell cystit (IC) är ett av dessa

sjukdomstillstånd, och kännetecknas av kronisk (långvarig)

inflammation, smärta, överaktivitet samt förtjockning av urinblåsan.

Kunskaper om vad som orsakar IC är begränsade, men man vet att IC

inte orsakas av en infektion. Flera studier har visat att urinblåsans

funktionalitet förändras vid cystit. Det övergripande målet med

denna avhandling var att förstå vilka skillnader som uppstår vid

inflammation i urinblåsan, och hur dessa påverkar urinblåsans

funktion. Fokus lades till stor del på urotelceller och hur dessa

förändras vid cystit. Känt sedan tidigare var att urotelceller har

förmågan att frisätta kvävemonoxid (NO). Avhandlingsarbetet

syftade till att klargöra hur denna frisättning påverkas vid cystit, och

vad NO-frisättningen har för påverkan på urinblåsan. Det var också

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känt sedan tidigare att en speciell sorts receptor, den muskarina M5

receptorn, uttrycks mer i urotelcellerna vid cystit. Vi ville ta reda på

om detta ökade receptoruttryck påverkar urinblåsans funktion.

För att framkalla ett tillstånd som efterliknar IC användes en

etablerad metod i vilken råttor injicerades med substansen

cyklofosfamid (CYP). Denna substans används inom sjukvården för

att behandla vissa former av cancer och svåra varianter av multipel

skleros (MS). En känd biverkning av denna behandling är

uppkomsten av cystit, och två dygn efter injektion med CYP

uppvisade 100% av försöksdjuren inflammation i urinblåsan. Denna

cystit är på flera sätt mycket lik den som finns hos patienter med IC.

Under studierna noterades konsekvent att lagret av urotelceller var

tjockare i inflammerade urinblåsor. Detta ledde till ett intresse att

undersöka hur celldelningen av urotelceller är reglerad. Specifikt

ville vi undersöka om det fanns någon receptor som var extra viktig

för denna celldelning. Dessa undersökningar gjordes på både friska

urotelceller och cancerurotelceller från människa.

Våra djurförsök visade att inflammation (cystit) orsakad av CYP

påverkar urinblåsans förmåga att kontrahera. Eftersom blockad av

frisättningen av NO från urotelceller vid cystit konsekvent

förbättrade urinblåsans förmåga att kontrahera drar vi slutsatsen att

NO utövar en dämpande effekt på funktionen i den inflammerade

urinblåsan. En minskad frisättning av NO var möjlig att åstadkomma

genom att antingen blockera bildningen av NO, blockera muskarina

receptorer eller helt enkelt ta bort urotelcellerna. Resultaten från

dessa studier bekräftade därmed att NO frisätts från urotelceller och

visade att denna frisättning kunde minskas genom att blockera

muskarina receptorer. Utifrån experimenten konstaterade vi att

denna muskarina receptor med ganska hög sannolikhet är av typen

M5. Om våra resultat är giltiga även för människa kan det innebära

en möjlighet att behandla den överaktivitet som uppstår hos den

inflammerade urinblåsan med ett läkemedel som selektivt påverkar

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den muskarina M5 receptorn, och därigenom påverkar frisättningen

av NO.

Våra försök på urotelceller indikerade att s.k. adrenoceptorer är

viktiga för regleringen av celldelning. Vi kunde se att aktivering av en

typ av adrenoceptor (β) ledde till ökad celldelning, medan aktivering

av en annan typ (α) ledde till minskad celldelning. Kolinerg

stimulering, t.ex. via muskarina receptorer, tycktes inte påverka

celldelningen.

En intressant fråga som ännu inte besvarats är exakt vilken roll (eller

roller) NO spelar under utvecklingen av cystit. Det kan vara så att NO

enbart är en produkt av inflammationen, eller så är NO en av de

molekyler som driver på utvecklingen av inflammationen. Det kan till

och med vara så att det är en kombination av båda dessa. Om NO är

med och driver på utvecklingen av inflammationen skulle det

teoretiskt sett vara möjligt att förhindra uppkomsten av cystit genom

att hämma bildningen av NO. Detta skulle visserligen kräva att man

utvecklar ett test för att upptäcka inflammationen när den är i sin

linda, men skulle potentiellt sett kunna minska lidandet för

miljontals människor världen över.

Sammanfattningsvis visar denna avhandling att råttans urinblåsa

förändras vid inflammation. Betydelsen av NO frisatt från

urotelceller ökar, och detta NO minskar urinblåsans förmåga att

kontrahera. Frisättningen sker, åtminstone till viss del, som svar på

aktivering av muskarina receptorer i urotelcellerna. Dessa

muskarina receptorer är med stor sannolikhet av typen M5. Framtida

studier bör göras för att avgöra om våra fynd är tillämpbara även på

människa. Framtida experiment bör också göras för att undersöka

betydelsen av NO vid utvecklingen av cystit.

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List of Publications

This thesis is based on the following papers, which are referred to by

their Roman numerals in the text:

I. Andersson MC., G. Tobin & D. Giglio, 2007

Cholinergic nitric oxide release from the urinary bladder

mucosa in cyclophosphamide-induced cystitis of the

anaesthetized rat

Br J Pharm 2008 Apr;153(7):1438-44. Epub 2008 Feb 4

II. Andersson M., P. Aronsson, D. Doufish, A. Lampert &

G. Tobin

The muscarinic M5 receptor is the primary mediator of

urothelium-derived nitric oxide effects in the rat urinary

bladder

Manuscript

III. Andersson M., P. Aronsson, D. Giglio, A. Wilhelmson, P.

Jeřábek & G. Tobin, 2010

Pharmacological modulation of the micturition pattern in

normal and cyclophosphamide-pretreated conscious rats

Auton Neurosci 2010. Epub 2010 Sep 17

IV. Andersson M., P. Aronsson, D. Eskandari, D. Giglio & G. Tobin

Characterization of receptor-mediated proliferation in the

human bladder urothelial UROtsa and T24 cell line

Manuscript

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Table of contents

ABSTRACT .............................................................................................................. 4

POPULÄRVETENSKAPLIG SAMMANFATTNING ........................................................ 6

LIST OF PUBLICATIONS ........................................................................................... 9

TABLE OF CONTENTS ............................................................................................ 10

LIST OF ABBREVIATIONS ....................................................................................... 12

INTRODUCTION .................................................................................................... 14

ANATOMY OF THE URINARY BLADDER ............................................................................. 14

Urothelial morphology ...................................................................................... 16

NORMAL BLADDER FUNCTION ....................................................................................... 17

The efferent nervous system ............................................................................. 20

The afferent nervous system ............................................................................. 25

The urothelium .................................................................................................. 26

Nitric oxide ........................................................................................................ 28

URINARY TRACT DISORDERS – DISEASES AND TREATMENTS ................................................. 29

OAB - Overactive bladder.................................................................................. 29

Acute and chronic incontinence ........................................................................ 30

Interstitial cystitis .............................................................................................. 31

Urinary bladder cancer ..................................................................................... 32

AIMS .................................................................................................................... 34

SPECIFIC AIMS ........................................................................................................... 34

METHODS AND MATERIALS .................................................................................. 35

CYCLOPHOSPHAMIDE-INDUCED CYSTITIS ......................................................................... 35

REMOVAL OF UROTHELIUM .......................................................................................... 35

CYSTOMETRICAL STUDIES ............................................................................................. 36

IN VITRO FUNCTIONAL STUDIES – THE ORGAN BATH ........................................................... 37

IN VIVO FUNCTIONAL STUDIES – THE METABOLISM CAGE .................................................... 38

CELL CULTIVATION ...................................................................................................... 39

MEASUREMENT OF PROLIFERATION ............................................................................... 39

MATERIALS ............................................................................................................... 40

Drugs ................................................................................................................. 40

Solutions and reagents ..................................................................................... 41

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STATISTICAL CALCULATIONS .......................................................................................... 41

Paper I ............................................................................................................... 41

Paper II .............................................................................................................. 42

Paper III ............................................................................................................. 42

Paper IV............................................................................................................. 42

RESULTS AND DISCUSSION ................................................................................... 44

PAPER I .................................................................................................................... 44

PAPER II ................................................................................................................... 48

PAPER III .................................................................................................................. 53

PAPER IV .................................................................................................................. 58

CONCLUDING REMARKS ....................................................................................... 63

ACKNOWLEDGEMENTS ......................................................................................... 67

REFERENCES ......................................................................................................... 69

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List of Abbreviations

4-DAMP 4-diphenylacetoxy-N-methylpiperidine

methobromide

AC adenylate cyclase

ACh acetylcholine

ANOVA analysis of variance

APF anti-proliferative factor

ATP adenosine 5’-triphosphate

BPH benign prostatic hyperplasia

BPS bladder pain syndrome

Ca2+ calcium

cAMP cyclic adenosine monophosphate

CYP cyclophosphamide

DAG diacylglycerol

DMEM Dulbecco´s modified Eagle medium

DO detrusor overactivity

EGF epidermal growth factor

eNOS endothelial nitric oxide synthase

HB-EGF heparin-binding epidermal growth factor-like

growth factor

IC interstitial cystitis

IL-6 interleukin-6

IP3 inositol triphosphate

L-748,337 N-[[3-[(2S)-2-Hydroxy-3-[[2-[4-

[(phenylsulfonyl)amino]phenyl]ethyl]amino]

propoxy]phenyl]methyl]-acetamide

L-NAME Nω-nitro-L-arginine methyl ester hydrochloride

L-NNA Nω-nitro-L-arginine

LUTD lower urinary tract dysfunctions

MeCh methacholine

MS multiple schlerosis

NA noradrenaline

NANC non-adrenergic, non-cholinergic

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NO nitric oxide

NOS nitric oxide synthase

OAB overactive bladder

PBS phosphate buffered saline

PEST penicillin/streptomycin

pFHHSiD p-fluoro-hexahydro-sila-diphenidol

hydrochloride

PIP2 phosphatidylinositol 4,5-bisphosphate

PLC phospholipase C

PPADS pyridoxalphosphate-6-azophenyl-2’,4’-disulfonic

acid

SR59230A 3-(2-Ethylphenoxy)-1-[[(1S)-1,2,3,4-

tetrahydronaphth-1-yl]amino]-(2S)-2-propanol

oxalate salt

UTP uridine 5’-triphosphate

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Introduction

Imagine life without the urinary bladder. Day in and day out it

enables us to store and dispose of numerous waste products filtered

from the blood. Without it, the urinary system would be

dysfunctional. For most people, the actions of the urinary bladder

passes unnoticed, until it malfunctions. In Sweden more than

400 000 people have daily symptoms of urinary incontinence, and

annually approximately 4.5% of all diagnosed cancer has its origin in

the urinary bladder. Many of the treatments for diseases affecting the

urinary bladder are suboptimal, rendering the need for greater

knowledge, in order to be able to generate improved treatments.

Anatomy of the urinary bladder

The urinary bladder is a hollow muscular organ situated in the

anterior part of the lower abdomen, resting on the pelvic floor

(Figure 1). Its basic functions are to store and void urine. The

maximum capacity of the urinary bladder varies interindividually,

but various sources say somewhere between 0.5 – 1 L in the healthy

bladder. Urine produced in the kidneys enters the bladder via the

two ureters. The entrances for these are situated on each side of the

posterior wall of the bladder, also known as the bladder base. The

empty bladder has a pyramidal shape, giving it, apart from the

posterior base, a superior side, two inferiolateral sides and an apex

(Figure 1).

Voiding is performed by contraction of the large smooth muscular

layer known as the detrusor. Urine thereby exits the bladder via the

urethra, which is situated in the most inferior part of the bladder

(Figure 2a). The involuntarily controlled internal sphincter keeps the

urine from leaking into the urethra and remains closed until the

detrusor contracts and the pressure inside the bladder, the

intravesical pressure, rises. The area surrounding the internal

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sphincter is known as the bladder neck, and its contractile properties

differ slightly from the rest of the detrusor, relaxing while the rest of

the detrusor contracts. Eventually, urine will be voided once the

external urethral sphincter, a circular band of skeletal muscle which

is under voluntary control, is opened.

Figure 1. Lateral view of the female lower abdomen. The pyramidal shaped

urinary bladder is situated in the anterior part of the lower abdomen, resting on the

pelvic floor. Adapted from http://en.wikipedia.org/wiki/Abdomen

The inner surface of the detrusor, the mucosa, is folded into rugae

when the bladder is empty. As the bladder fills up, the surface turns

smooth. However, one area remains smooth at all times, the trigone.

This is a triangular shaped area of the detrusor, situated between the

openings of the two ureters and the urethral opening. The mucosa in

this area is thicker, and the population of receptors in the smooth

muscle has been shown to slightly differ from the rest of the detrusor

(Saito et al., 1997).

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Figure 2. Anatomy of the urinary bladder. (a) Anterior view of the urinary

bladder. Urine enters the urinary bladder via the ureters, and exits via the urethra.

(b) Cross-section of the bladder wall. Reprinted with permission (Absolute Medical, 2010).

Urothelial morphology

From the inside and outwards, the bladder consists of a mucosal

layer known as the urothelium, a suburothelium, three muscular

layers and a cover of serosa (Figure 2b). The three smooth muscle

layers consist of a circular muscle layer interlaced between two

longitudinal muscle layers. The urothelium is a stratified epithelium

consisting of 3-6 cell layers; a basal layer, one or more intermediate

layers and a surface layer (Jost et al., 1989). The size of the urothelial

cells increase from the base to the surface; the smallest being the

basal cells, intermediate cells being intermediate also in size, and

surface cells being the largest. Surface urothelial cells, often referred

to as umbrella cells due to their hexagonal umbrella-like

morphology, are laterally connected by tight junctions (Apodaca,

2004). Together with a cover of glycosaminoglycan and crystalline

proteins, uroplakins, these tight junctions maintain a barrier

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between the urine and the bloodstream (Parsons et al., 1990; Hu et

al., 2002; Born et al., 2003). Immediately beneath the urothelium one

finds a thin basal lamina, separating the urothelium from the lamina

propria. The lamina propria is today often referred to as part of the

suburothelium. The suburothelium consists of connective tissue

(bundles of collagen and elastic fibers), muscular bundles, afferent

and efferent nerve fibers and a numerous amount of capillaries.

Thus, the urothelium is not vascularized nor directly innervated by

the nervous system, but is still in close vicinity to capillaries and

nerve endings. Within the suburothelium one also finds

myofibroblasts. While the function of these cells has not been

entirely established, it has been suggested that they function much in

the same way as cells of Cajal do in the gut, modulating

neurotransmission and bladder function (Wiseman et al., 2003;

Tobin et al., 2009).

Normal bladder function

The urinary bladder has two basic functions; storage of urine and

emptying of urine. Since the production of urine is constantly active,

at all given times one of these functions are active. One often refers to

the urinary bladder being in either the filling or the emptying phase.

The sensations during the filling phase can be divided into three

distinct happenings; initial sensation of filling, first desire to void and

strong desire to void. During the filling phase, there is a concomitant

increasing contraction of the bladder outlet region (bladder neck and

urethra), and increasing relaxation of the detrusor. At the same time,

muscles and connective fibers in the pelvic floor help maintain

continence (Sampselle & DeLancey, 1998). The increasing relaxation

of the detrusor as the bladder fills up yields a situation where despite

bladder filling, the intravesical pressure remains rather constant.

However, the bladder cannot fill up forever and eventually the

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intravesical pressure will increase. As the bladder continues to fill up,

the balance is shifted towards decreased contraction of the bladder

outlet region, and increased contraction of the detrusor. Afferent

pressure sensing systems (stretch receptors) are activated,

rendering awareness to us that the bladder needs to be emptied.

Before we can empty the bladder the outer sphincter has to be

opened. This sphincter is via the pudendal nerve under our voluntary

control, at least in healthy individuals. Our voluntary opening of the

outer sphincter initiates the micturition reflex, causing the bladder

outlet region to relax and the detrusor to contract.

Like most organs in the human body, the urinary bladder is

innervated by both sympathetic and parasympathetic nerve fibers.

Activation of these nerve fibers leads to activation of muscarinic and

adrenergic receptors in the detrusor, respectively. The muscarinic

receptor population in the urinary bladder consists of five subtypes,

denoted M1-M5 (Caulfield & Birdsall, 1998; Giglio & Tobin, 2009).

Several studies have shown that M2 is the most abundantly

expressed subtype in the detrusor, with roughly a 3:1 ratio over the

functionally most important muscarinic receptor, the M3 receptor

(Eglen et al., 1994; Wang et al., 1995; Yamaguchi et al., 1996; Hegde

& Eglen, 1999; Sigala et al., 2002). The overall distribution of

muscarinic receptors varies throughout the urinary bladder, with a

generally lower concentration in the bladder neck (Saito et al., 1997).

Three of the muscarinic receptor subtypes, namely the M1, M3 and

M5 receptor, are Gq/11 coupled receptors whose activation induces

the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to

diacylglycerol (DAG) and inositol triphosphate (IP3) by

phospholipase C (PLC; Figure 3). Both products (DAG and IP3)

increase the amount of intracellular calcium (Ca2+). In the detrusor

this results in an increased tonus. The other two subtypes are Gi

coupled, and even if several pathways have been suggested (Bolton &

Zholos, 1997; Togashi et al., 1998), in man they predominantly

inhibit adenylate cyclase (AC) upon activation (Figure 3).

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Figure 3. Main intracellular pathways of muscarinic and β-adrenergic

receptors in the urinary bladder. An increase of intracellular calcium causes an

increase in detrusor tonus, while an increase of intracellular cAMP causes a decrease

in detrusor tonus. The increase of intracellular calcium upon muscarinic M1/M3/M5

receptor activation is achieved through two separate mechanisms; (1) diacylglycerol

(DAG) in the cell membrane causes an influx via L-type Ca2+ channels and (2)

calcium is released from the sarcoplasmatic reticulum (SR) upon inositol

triphosphate (IP3) stimulation.

The adrenergic receptors, or adrenoceptors, which are widespread

throughout the lower urinary tract, are divided into the α- and β-

adrenoceptors. The α-adrenoceptors are further subdivided into the

α1- (α1A, α1B and α1D) and α2-(α2A, α2B and α2C)adrenoceptors. The β-

adrenoceptors are divided in to three subtypes; the β1-, β2- and β3-

adrenoceptor. All subtypes are found in the urinary bladder of both

man and rat, predominantly in the bladder body (Levin et al., 1988;

Seguchi et al., 1998). One study determined the β3 subtype to be the

most abundant in the human bladder in regard to mRNA expression

(Nomiya & Yamaguchi, 2003). Activation of β-adrenoceptors mainly

20

leads to the formation of cyclic adenosine phosphate (cAMP), and the

subsequent lowering of detrusor tonus (Figure 3).

Figure 4. Autonomic innervation of the male urinary bladder, prostate gland

and urethra. The sympathetic nervous system innervates the urinary bladder and

prostate gland mainly via the hypogastric nerve. The parasympathetic nervous

system innervates the urinary bladder mainly via the pelvic nerve. The external

urethral sphincter, which is under voluntary control, is primarily innervated by the

pudendal nerve.

The efferent nervous system

Sympathetic and parasympathetic nerve fibers reach the urinary

bladder mainly via the hypogastric and pelvic nerve, respectively

(Figure 4). During the filling phase, it is predominantly the

sympathetic nerve fibers in the hypogastric nerve that are active,

maintaining tonus in the bladder neck to avoid unwilling leakage of

urine. At the same time, centers in the brain and brainstem act to

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suppress the parasympathetic activity. When the threshold is

reached, and we have willingly opened the outer sphincter, the

balance between sympathetic and parasympathetic activation is

reversed. Then the pelvic nerve signals dominate, causing a

contraction of the detrusor and a relaxation of the bladder neck and

urethra.

During the filling phase, several receptor subtypes are involved in

the maintaining of constant intravesical pressure. Adrenergic α1-

receptors, particularly of the α1A-subtype, are dominant in the

bladder neck region, causing contraction of the smooth muscle

(Caine et al., 1975; Andersson et al., 1984; Walden et al., 1997; Chang

et al., 2000). Partly therefore, α1 antagonists are used to relieve outlet

obstruction symptoms in elderly men with benign prostatic

hyperplasia (BPH). However, α1-adrenoceptors are poorly expressed

in the rest of the detrusor (Goepel et al., 1997; Malloy et al., 1998;

Sigala et al., 2004), and their physiological relevance there seems to

be of low importance (Ueda et al., 1984; Lluel et al., 2003). Instead, β-

adrenoceptors are responsible for relaxation of the detrusor during

the filling phase (Figure 5; Elmer, 1974; Nergardh et al., 1977; Abdel-

Rahman et al., 1983). In man, it is predominantly the β3-

adrenoceptor that mediates relaxation, while in rat it is a

combination of β2- and β3-adrenoceptor activation (Oshita et al.,

1997; Igawa et al., 1998; Yamazaki et al., 1998; Igawa et al., 2001).

Studies have shown that β-adrenergic tone increases with increasing

intravesical pressure (Lecci et al., 1998) and that the nerve fibers

that mediate this relaxation are sensitive to both prostaglandins,

neurokinin A and capsaicin (Tucci et al., 2002).

Noradrenaline (NA) is the key signal substance of efferent

sympathetic innervation. Upon activation, the release of NA can be

modulated by several mechanisms, including negative feedback on

pre-junctional α2-adrenoceptors (Mattiasson et al., 1987; Somogyi &

de Groat, 1990). However, in some species such as pig, cat and

22

guinea-pig, bladder smooth muscle seems to lack expression of α2-

adrenoceptors, and it is unclear whether α2 activation affects bladder

function other than via blood flow regulation (Ueda et al., 1984;

Monneron et al., 2000). In rat, the release of NA can also be facilitated

by both activation of muscarinic M1 receptors as well as α1-

adrenoceptors (Somogyi et al., 1995; Somogyi et al., 1996).

Figure 5. Depiction of the nervous control of detrusor tonus. Activation of the

sympathetic nervous system leads to relaxation of the detrusor, which is mediated

mainly via activation of -adrenergic receptors. Activation of the parasympathetic

nervous system leads to contraction (increased tonus) of the detrusor, which is

mediated directly via muscarinic M3 receptors and indirectly via muscarinic M2

receptors.

The primary signal substance released by efferent parasympathetic

nerves, activating muscarinic receptors throughout the urinary

23

bladder, is acetylcholine (ACh). Binding of ACh to muscarinic

receptors in the detrusor causes it to contract, rendering the bladder

to empty. Even though it is the M3 receptor that is considered the

functionally most important subtype for detrusor contraction, it is

thought that there is a dual effect between the activation of M3 and

M2 receptors (Figure 5; Hegde & Eglen, 1999). In the healthy human

bladder, the M3 subtype has been shown in functional studies to be

responsible for direct contraction (Chess-Williams et al., 2001;

Fetscher et al., 2002). Second messenger studies have confirmed

these results, showing an increased formation of IP3 upon M3

activation (Noronha-Blob et al., 1989; Andersson et al., 1991). Also,

studies on M3 knock-out mice revealed a 95% reduction in bladder

contractile response to carbachol (Matsui et al., 2000).

Activation of M2 receptors has been suggested to yield an indirect

contraction by inhibiting β-adrenoceptor and purinergic receptor

mediated relaxation (Hegde et al., 1997; Giglio et al., 2001; Chess-

Williams, 2002; Giglio et al., 2005a). These findings are supported by

second messenger studies that show a decrease of cAMP upon

carbachol stimulation (Noronha-Blob et al., 1989; Harriss et al.,

1995). Even though it has never been shown in man that M2 plays a

significant role in the direct contraction of the healthy urinary

bladder, some studies indicate an increased role of M2-mediated

detrusor contraction during disease (Eglen et al., 1994; Braverman et

al., 1999).

The release of ACh from parasympathetic nerve endings can be both

inhibited and facilitated via activation of different muscarinic

receptors. Prejunctional M1 receptors have been shown to facilitate

the release of ACh in several species (Somogyi et al., 1994; Tobin &

Sjogren, 1995; Inadome et al., 1998). It has also been shown that this

facilitatory effect can increase during hyperreactive disease states

(Somogyi et al., 1998). Which prejunctional muscarinic receptor that

inhibits ACh release via negative feedback seems to vary among

24

species (for review see Somogyi et al. (1999)). Prejunctional M2/M4

receptors have been found to inhibit the release of ACh in rat and

rabbit (Tobin, 1995; Tobin & Sjogren, 1995; Braverman et al., 1998),

and later studies indicated that M4 receptors do the same in rat and

man (D'Agostino et al., 1997; D'Agostino et al., 2000). Apart from

muscarinic receptors, also the activation of α1- and α2-adrenoceptors

has been shown to facilitate and inhibit the release of ACh,

respectively (Nakamura et al., 1984; Somogyi et al., 1995; Tobin &

Sjogren, 1998; Szell et al., 2000).

In several species it has been shown that there can be other signal

substances than ACh that are involved in bladder emptying. In cat

and rat for instance, adenosine 5’-triphosphate (ATP) is co-released

together with ACh from parasympathetic nerve fibers (MacKenzie et

al., 1982; Chancellor et al., 1992; Palea et al., 1993; Theobald, 1995).

In these species ATP acts upon P2X1 receptors (Aronsson et al.,

2010), causing a fast transient contraction which possibly is the

initiator of the detrusor contraction (for review see Burnstock,

2009). The co-release of ATP makes up the most important part of

the so called atropine-resistant component of parasympathetically

induced bladder contraction, a part that hence cannot be blocked by

the non-selective muscarinic antagonist atropine. In man, even

though it has been hard to show that the atropine-resistant

component is of any great importance in the healthy bladder, ATP

can play a role in the activation of the detrusor during certain disease

states and in the aged bladder (Sjogren et al., 1982; Bayliss et al.,

1999; Yoshida et al., 2001). On the other hand, the atropine-resistant

component in the healthy bladder has been shown to be of greater

importance in both rabbit (Longhurst et al., 1984), guinea-pig

(Chesher & Thorp, 1965), cat (Theobald, 1996) and rat (Giglio et al.,

2007).

25

The afferent nervous system

Several afferent systems in the urinary bladder work together in

order to maintain optimal bladder function. Afferent, or sensory,

nerve fibers are widespread throughout the detrusor, some which

extend into the basal layer of the urothelium (Andersson, 2002a).

These nerve fibers are mainly conveyed via the pelvic nerves, but

also via the hypogastric and pudendal nerve (Andersson, 2002a). The

sensory nerves involved are both of the myelinated Aδ-fiber and

unmyelinated C-fiber type. Roughly, the Aδ-fibers convey sensation

of filling while the C-fibers mediate thermal stimulus and relay

information of chemical irritation (Janig & Morrison, 1986; Fall et al.,

1990; Habler et al., 1990). Stretch receptors in the bladder wall are

stimulated by increased intravesical pressure. These receptors

activate the Aδ afferent nerve fibers in the pelvic nerve that carry the

signal to the sacral spinal cord. The afferent signaling can be

modulated by several transmitters, including ATP (Andersson,

2002b).

Adrenergic α1A-receptors found in the urothelium have been shown

to modulate bladder afferent activity in rats, shortening the

intercontraction interval (Yanase et al., 2008). The physiological

relevance of this finding is debatable, but perhaps valid during

certain patophysiological conditions that can give increased

concentrations of NA in the urine. Several other substances have also

been shown to be able to modulate afferent activity including

neutrophins, prostaglandins and tachykinins (Yoshimura & de Groat,

1997). While prostaglandins mainly seem to act as sensitizers of

afferents, intravesically administered tachykinins are able to cause

direct contraction of the detrusor (Ishizuka et al., 1995a, b).

26

The urothelium

For a long time the urothelium was considered solely a protective

barrier, shielding the urinary bladder, as well as the rest of the body,

from the toxic and corrosive urine. But during the last decade several

studies have showed that the urothelium does much more than so.

For instance, the urothelium has been shown to respond to its

environment in regard to luminal pressure and urine composition by

releasing diffusible agents such as acetylcholine (ACh), ATP and

nitric oxide (NO) (Ferguson et al., 1997; Klapproth et al., 1997; de

Groat, 2004). To exemplify, the mechanism of stretch-induced ATP

release from the basolateral side of urothelial cells is well established

(Ferguson et al., 1997). It has also been shown that the release of

ATP can increase upon infection (Save & Persson, 2010). This

release, and that of other diffusible agents, can in turn affect afferent

nerves and smooth muscle bundles in the suburothelium, thereby

assigning the urothelium modulatory properties. Within the

suburothelium one finds nerve fibers reactive to cholinergic,

adrenergic, purinergic (P2X) and peptidergic stimuli (Jen et al.,

1995). Since receptor stimulation on some of these nerves has been

shown to evoke exocytosis, they could be both of an afferent and

paracrine/modulatory nature at the same time. Interestingly, the

expression and function of the diffusible agents from the urothelium

that can affect these nerves can be altered by urinary bladder

diseases such as cystitis (Sun et al., 2001; Giglio et al., 2005b; Smith

et al., 2005). Further, the urothelium is capable of directly

responding to various stresses, such as ischemia and urinary tract

infections (Mysorekar et al., 2002).

27

Receptor Activator Method of detection

References

Muscarinic (M1-M5)

ACh

IHC, IB RT-PCR

Giglio et al., 2005 Tyagi et al., 2006

Nicotinic (subunits alpha 3, 5, 7, 9, 10 and beta 3, 4)

ACh RT-PCR Beckel et al., 2006 Bschleipfer et al.,

2007

Purinergic (P1A1, P1A2A, P1A2B, P1A3)

adenosine IB Yu et al., 2006 Säve et al., 2009

Purinergic (P2X2,3 and P2Y1,2,4)

ATP ICC, IB, PCR RT-PCR

Chopra et al., 2008 Tempest et al., 2004

Adrenergic (α1A, α1D and β1-3)

A/NA IB, IHC RT-PCR

ISH

Ishihama et al., 2006 Otsuka et al., 2008 Walden et al., 1997

PAC1, VPAC1, VPAC2 VIP/PACAP ICC RT-PCR

Braas et al., 2006 Girard et al., 2008

EGFR EGF IHC Røtterud et al., 2005

Tachykinin (NK1) substance P RT-PCR, IB IHC

Sanchez Freire et al., 2010

Vanilloid (TRPV; VR1) pH, heat, capsaicin RT-PCR, ICC

Birder et al., 2001

Bradykinin (B2) bradykinin RT-PCR, ICC

Chopra et al., 2005

Table 1. Receptor expression in urothelial cells. ACh - acetylcholine; ATP -

adenosine 5’-triphosphate; A - adrenaline; NA - noradrenaline; VIP - vasointestinal

peptide; PACAP - pituitary adenylate cyclase activating polypeptide; EGF - epidermal

growth factor; RT-PCR - reverse transcriptase polymerase chain reaction; IHC -

immunohistochemistry; IB - immunoblotting; ICC - immunocytochemistry; ISH - in

situ hybridization.

28

All five muscarinic receptor subtypes have been shown to be present

in both human and rat urothelium (Giglio et al., 2005b; Tyagi et al.,

2006). Activation of these has been shown to lead to a number of

events, showing that they possess functional properties. For instance,

in both man and pig an unidentified relaxant factor has been shown

to be released from the urothelium upon muscarinic receptor

activation (Hawthorn et al., 2000; Chaiyaprasithi et al., 2003). Others

have shown that activation of urothelial muscarinic receptors can

lead to the release of ATP, which possibly modifies the bladders

afferent response (Kullmann et al., 2008a; Kullmann et al., 2008b;

Munoz et al., 2010). In turn, ATP and uridine 5’-triphosphate (UTP)

have been shown to yield larger contractions in intact than

urothelium-denuded strips of guinea-pig detrusor (Sui et al., 2008),

further demonstrating the presence of functional urothelial

receptors. The most likely candidates for this functional response are

of the P2X subtype, but also the presence of P1 and P2Y receptors

has been shown in the urothelium (Tempest et al., 2004; Yu et al.,

2006; Chopra et al., 2008). Apart from muscarinic and purinergic

receptors, urothelial cells express a vast array of receptors (see

Table 1).

Nitric oxide

Nitric oxide (NO) is generally accepted as one of the non-adrenergic,

non-cholinergic (NANC)-transmittors affecting the bladder (Bult et

al., 1990; Andersson & Persson, 1994). Studies using a NO scavenger

have shown that NO can be involved in normal bladder activity, and

that the removal of NO can cause a condition similar to overactive

bladder (Pandita et al., 2000). During conditions such as

cyclophosphamide (CYP)-induced cystitis, nitric oxide synthase

(NOS) has been shown to be up-regulated (Alfieri & Cubeddu, 2000;

Giglio et al., 2005b), and NO has been suggested to not only affect the

detrusor, but also act directly on afferent pathways (Ozawa et al.,

1999; Yoshimura et al., 2001).

29

NO can be released in the bladder in several ways. Studies have

shown the presence of nitric oxide containing nerve fibers in the

suburothelium, but their exact function is still unclear (Smet et al.,

1996). Others have seen release of NO from cultured urothelial cells

in response to muscarinic agonists (Kullmann et al., 2008b), and

from urinary bladder strips in response to adrenergic agonists

(Birder et al., 1998). A recent study by Munoz et al. (2010) could

show the release of NO from the urothelium upon muscarinic

receptor stimulation, but also other parts of the rat urinary bladder

could release NO, most likely the suburothelium.

Urinary tract disorders – diseases and treatments

OAB - Overactive bladder

One of many lower urinary tract dysfunctions (LUTD) is overactive

bladder (OAB), defined as urgency, with or without incontinence,

usually accompanied by increased frequency and nocturia (Abrams et

al., 2002). For a patient to be diagnosed with OAB, a concomitant

infection has to be ruled out. The prevalence of OAB is about 17%

(Milsom et al., 2001), but few prospective studies have been

performed. The cause of OAB is not well understood, but the general

risk of developing OAB increases with age. One possible cause of this

has been shown in a study by Li et al. (2003) where they found the

amount of binding sites for β-adrenoceptor agonists to decrease with

age. Since it has been shown that the most important function of β-

adrenoceptors is mediating relaxation of the detrusor during bladder

filling, it would be logical that a decrease of sympathetic activity

yields an increase in detrusor sensitivity. However, the most

widespread theory is increased afferent activity, either depending on

increased sensitivity or an increased number of afferents (Drake,

2008).

30

β3-agonists, due to their hampering of bladder contraction, have been

suggested to be a possible treatment for OAB, but to date no such

drug has been successfully developed. During the testing of

experimental β3-agonists on rats, induced cystitis and renal tubular

necrosis was reported (Waghe et al., 1999). Since the beneficial site

of action very well may be at the spinal level (Durant et al., 1988),

intrathecal administration could be a possible route of

administration, but that is something that most patients would

probably wish to avoid. However, the adverse effects seem to vary

considerably between different substances, and some promising oral

β3 agonist candidates are currently in phase II or phase III trials (for

review see Drake, 2008).

Despite the promising future, the drug treatments available today are

few. Patients are advised to manage their fluid intake and avoid

certain drinks, for instance those containing caffeine. The by far most

common drug treatment is antimuscarinic drugs, which give good

clinical effect initially, but regular long-term loss of function and

compliance problems (Andersson, 2009). Apart from the

antimuscarinic drugs and β3-adrenoceptor agonists, Ca2+-antagonists

and K+-channel openers have been suggested as possible inhibitors

of detrusor activity (Guarneri et al., 1991; Pandita et al., 2006). Also,

drugs that reduce afferent activity and/or affect NK-1 receptors at

the spinal level are under development and are currently in clinical

phase II or III trials (for review see Drake, 2008).

Acute and chronic incontinence

Urinary incontinence, or enuresis, is defined as involuntary leakage of

urine (Abrams et al., 2002). This involuntary leakage can either be a

constant dribbling or large emptying of the bladder. The large

emptying can be caused by stresses such as sneezing or coughing, or

be so called urge incontinence, a condition where the patient feels

the urge to micturate, but does not have time to reach the toilet

31

before the expel of urine. While stress incontinence is by far the most

common form in women (80%), in men urge incontinence is the

most widespread form (Hampel et al., 2004). Incontinence in any

form is a very common condition, and prevalence increases with

increasing age. It has been estimated that 250 million people

worldwide suffer from incontinence, and in Sweden the prevalence

among 80-year olds is as high as 25% (Fall, 1999; Milsom, 2009).

Treatment of acute and chronic incontinence has, apart from lifestyle

changes, focused on antimuscarinic drugs. Older drugs, such as

oxybutynin, are basically unselective, whereas the development of

new treatments has focused on finding M3-selective compounds.

However, no drug has yet been launched that does not cause the

most common adverse effects; dry mouth and gastrointestinal

disturbances. The main reason for that is surely due to the fact that

these adverse effects per se are M3 receptor-mediated (Tobin, 1995;

Barras et al., 1999). The mechanism of action of antimuscarinic drugs

was first thought to be solely by blocking efferent muscarinic

receptors in the detrusor. But further studies have shown that both

the urothelium and afferent nerve fibers are possible sites of action

(Kim et al., 2005; Abrams et al., 2006; Yamaguchi, 2010).

Other possible treatments include β agonists. One study found that in

bladder strips taken from patients with urge incontinence,

clenbuterol, a fairly β2-selective agonist, gave a significantly greater

relaxation than in bladder strips taken from patients without any

history of incontinence (Hudman et al., 2001). This finding in itself is

interesting, but as previously discussed, it is mainly the β3-

adrenoceptor that mediates relaxation in man.

Interstitial cystitis

Interstitial cystitis (IC), which is also known as bladder pain

syndrome (BPS), is a non-infectious chronic pain syndrome that is

32

characterized by painful urination, urgency, frequency, inflammation,

stiffening of the bladder wall and visceral pain. In its classic form one

finds ulcers upon cytoscopic examination, but in most cases (~80%)

the disease is ulcer-free (Koziol et al., 1996). As the symptoms are

similar to other LUTD, and the objective diagnostic criteria are few,

the epidemiology of IC is difficult to determine (Hanno, 2002).

Estimations vary between 10 per 100 000 to 70 per 100 000

(Oravisto, 1975; Curhan et al., 1999). The etiology of IC is mostly

unknown, and treatment regimens are mainly symptomatic.

However, sensitization of afferent pathways, as well as altered

release of ATP and NO, has been linked to the condition (Sun et al.,

2001; Logadottir et al., 2004). Further, the expression of muscarinic

receptors has been shown to be up-regulated, and studies have

linked the increase of certain inflammatory markers (mainly IL-6)

and changes in the levels of proliferative factors such as anti-

proliferative factor (APF), epidermal growth factor (EGF) and

heparin-binding epidermal growth factor-like growth factor

(HB-EGF) in urine to the condition (Keay et al., 1997; Erickson et al.,

2002; Sanchez Freire et al., 2010).

Urinary bladder cancer

Urinary bladder cancer accounts for 4-5% of all diagnosed cancer in

Sweden, making it the 6th most common form of cancer (Cancer

Incidence in Sweden 2007, Socialstyrelsen, 2008; IARC Globocan,

2008). Worldwide it is the 11th most common form of cancer,

accounting for about 3% of all reported cancers (IARC Globocan,

2008). However, the worldwide estimation is probably somewhat on

the small side as several developing countries, with a lower

probability of reporting, have high incidences of Bilharzia (also

known as Schistosomiasis) infections, a known cause of bladder

cancer (Elsebai, 1977; Schwartz, 1981).

The major causative agents for bladder cancer are various chemical

33

carcinogens, with different metabolites from cigarette smoking

accounting for about half of the cases (Shirai, 1993). More

particularly, these carcinogenic metabolites are thought to

accumulate in the urine, irritate certain urothelial cells to the degree

that they develop point mutations, and eventually cause them to

transform (for review see Brandt, 2009). As a result of this, most

urinary bladder cancer (~90%) has its origin in the urothelium

(Cordon-Cardo, 2008). Chemical carcinogens aside, also chronic

inflammation, caused by infections, indwelling catheters or

pharmacological treatment, has been shown to increase the risk of

developing bladder cancer (for review see Michaud, 2007). Studies

on Bilharzia, both clinical and pre-clinical, have shown a chain of

events starting with infection, continuing with chronic inflammation

and eventually leading to bladder cancer (Samaras et al., 2010). The

coupling between inflammation and cancer is well-established and

has been shown in several other tissues (Coussens & Werb, 2002;

Ohshima et al., 2003). Apart from Bilharzia infection, it has also been

shown that long-term treatment (>10 years) with CYP, generating a

state similar to chronic inflammation, increases the risk of

developing bladder cancer (Travis et al., 1995; Talar-Williams et al.,

1996).

Presently, the exact mechanisms of transformation of urothelial cells

from healthy to malignant are incompletely understood, and

therefore we today only have treatment options for already arisen

tumors. In the future we hopefully will have greater knowledge of the

transformational mechanisms, giving us the opportunity to develop

treatment strategies that prevent the origination of bladder cancer.

34

Aims

The main aim of this thesis was to investigate the effects of CYP pre-

treatment in rats, with special interest in the release of NO from the

urothelium.

Specific aims

In vivo in the anaesthetized rat (Paper I), we wondered how CYP pre-

treatment affected the muscarinic bladder contractile responses and

if NO might be released from the urothelium by cholinergic stimuli.

Our studies confirmed that the muscarinic bladder contractile

responses were altered by CYP pre-treatment, and our data indicated

that one component of this alteration was regulated by the

cholinergic release of NO from the urothelium. This led us to a study

in vitro on bladder strips (Paper II) where we wanted to characterize

the muscarinic changes involved in the alterations during CYP-

induced cystitis. This study confirmed the results found in vivo and

raised the question whether or not CYP pre-treatment altered the

micturition parameters in the conscious rat and, further, if

muscarinic, purinergic or NOS blockade could alter these changes

(Paper III). In all studies on CYP pre-treated rats, we could see

morphological changes, most prominently an increase in the

thickness of the bladder wall. Several studies have shown a coupling

between chronic CYP-induced cystitis, including bladder wall

thickening, and the development of urothelial bladder cancer. This

led us to wonder how the proliferation of urothelial cells is regulated

(Paper IV); more specifically, if the proliferation rate of healthy and

malignant urothelial cells is receptor mediated and if there are any

changes in this regulation after transformation.

35

Methods and Materials

All experiments conducted during this thesis were approved by the

local ethics committee at the University of Gothenburg. Consistently,

male rats of the Sprague-Dawley (300 – 400 g) strain were used.

Cyclophosphamide-induced cystitis

Cyclophosphamide (CYP)-induced cystitis is a common and well

documented method for inducing experimental cystitis (deVries &

Freiha, 1990). CYP is a DNA alkylating agent used in man to treat

neoplastic diseases and multiple schlerosis. Its metabolite acrolein

accumulates in the bladder lumen, where it causes severe irritation

of the urothelium, subsequently inducing haemorrhagic cystitis (Cox,

1979). To induce cystitis we have given a single intraperitoneal dose

of CYP (100 mg/kg) in combination with the analgesic

buprenophinum (Temgesic®; 10 g/kg). As previously seen in our

lab, the peak inflammatory state arises after 60 h (Giglio et al.,

2005b). Depending on which type of experiment was to be carried

out, we waited for an appropriate time before we conducted the

experiments. I.e., for cystometrical studies and in vitro functional

studies in the organ bath, experiments were carried out 60 h after

induction of cystitis. For in vivo functional studies in the metabolism

cage, in which the experiments were conducted during a 24 h period,

the experiments were started 48 h after induction of cystitis and thus

continued until 72 h post induction, comprising the 60 h peak

inflammatory time point.

Removal of urothelium

To study the direct impact of the urothelium on bladder function we

conducted both in vivo and in vitro experiments where the

urothelium was removed. In order to achieve this, we injected

36

collagenase (0.1% in Hank’s balanced salt solution) directly into the

bladder of an anaesthetized rat, either via a secured catheter or

needle. The volume injected varied between 0.2 - 0.5 mL, depending

on the size of the bladder. Careful notice was taken to first empty the

bladder of urine, and then properly fill it without distending it

extensively. Once injected, the collagenase was left in the bladder for

30 min, and then removed via the catheter/needle. Thereafter, the

serosal side of the bladder was gently rubbed, and loose urothelial

cells were removed by rinsing the bladder with saline. Regularly

after the conduction of the experiments, the bladder was

microscopically examined in order to ensure that the urothelium was

properly removed.

Cystometrical studies

Rats (300 - 350 g) were anaesthetized with an intraperitoneal

injection of pentobarbitone (45 mg/kg). During the remainder of the

experiment supplementary doses were given intravenously when

needed. The rats were placed on a thermostatically controlled

blanket, keeping their body temperature at 38°C. Airways were kept

free by tracheotomy and placement of a cannula in their trachea.

Catheters were placed in both the femoral artery and femoral vein, in

order to monitor blood pressure and administer drugs, respectively.

Two cannulas were inserted through a small incision at the apex of

the bladder, where they also were fixed to the bladder with a

ligature. One of the bladder cannulas was used to maintain bladder

pressure at 10 - 15 mmHg at all times by injection of small volumes

of saline (0.05 - 0.20 mL), and the other was used to monitor

intravesical pressure. When needed, the same cannula that was used

to maintain intravesical pressure was also used to instill collagenase.

Both the catheter in the femoral artery and the pressure sensing

cannula in the urinary bladder were connected to pressure

transducers, as part of the MP100WSW data acquisition system. All

data was recorded using the Acquire software (Biopac, Goleta, USA).

37

Adrenergic effects were blocked in all experiments by administration

of the phentolamine (1 mg/kg i.v.) and propranolol (1 mg/kg i.v.). At

the beginning of each experiment, regardless of series, the

unselective muscarinic agonist methacholine (MeCh) was given in

increasing doses (0.5 – 5 µg/kg) in order to establish a dose-

response curve. Dose-response curves were therefore generated for

untreated control rats and CYP pre-treated rats, with and without

urothelium. In one experimental series, dose-response curves were

also generated for CYP pre-treated rats, before and after removal of

the urothelium, in the presence of the NOS inhibitor

Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME; 30 mg/kg

i.v.). In another experimental series, L-NAME (30 mg/kg i.v.) or saline

(0.9% i.v.) was given to normal rats with an intact urothelium and

CYP pre-treated rats with or without urothelium. Thereafter,

repeated doses of MeCh (2 µg/kg i.v.) were given after increasing

doses of the muscarinic M1/M3/M5-selective antagonist

4-diphenylacetoxy-N-methyl-piperidine methobromide (4-DAMP;

0.1 – 1000 µg/kg i.v.). After each experiment, rats were killed with an

overdose pentobarbitone and their urinary bladders were examined

macroscopically and microscopically.

In vitro functional studies – the organ bath

Experiments on detrusor contractility were conducted in an organ

bath setup. Rats (300 - 350 g; n=52), either CYP(100 mg/kg i.p.) or

saline (0.9%; 1 mL/kg i.p.; serving as controls) pre-treated, were

anaesthetized with an intraperitoneal injection of medetomidine

(Domitor®; 1 mg/kg). Thereafter, they were killed with an overdose

of carbon dioxide and the urinary bladder, with an intact urothelium

or after removal of the urothelium, was removed and placed in Krebs

bicarbonate solution. Via a cut along the posterior surface from the

urethral opening to the apex the bladder was opened, and two or

three full thickness strips (6 x 2 mm) were excised from the detrusor

38

medially to the orifices of the two ureters. The detrusor strips were

mounted between two steel rods, of which one was fixed and the

other was adjustable and connected to an isometric force transducer

(Linton). Thereafter, they were immersed in 25 mL organ baths

containing Krebs bicarbonate solution of the following composition

(mM): NaCl 118, KCl 4.6, CaCl2 1.25, KH2PO4 1.15, MgSO4 1.15,

NaHCO3 25, and glucose 5.5. The baths were kept at 37°C and gassed

with 5% CO2 in O2. The strips were repeatedly stretched for 45 min,

in order to achieve a stable tension of about 5 mN. At the beginning

of each experiment, a high K+ Krebs solution (containing 124 mM K+;

obtained by exchanging Na+ for equimolar amounts of K+) was

administered in order to assess the viability of each strip

preparation. All antagonists, as well as the NOS inhibitor

Nω-nitro-L-arginine (L-NNA), were administered 20 min before each

series of the unselective muscarinic agonist carbachol. All drugs were

administered to the organ baths at a volume of 125 µl. Data were

recorded using a MP100WSW data acquisition system and the

Acquire software (Biopac, Goleta, USA).

In vivo functional studies – the metabolism cage

For these experiments, rats (310 - 400 g; n=100) were pre-treated

with either saline (0.9%; 1 mL/kg i.p.; serving as controls) or CYP

(100 mg/kg i.p.), both in combination with the analgesic

buprenorphinum (10 µg/kg i.p.). Forty-eight hours later, they were

injected intraperitoneally with either saline (0.9%), 4-DAMP

(1 mg/kg), the purinergic P2 receptor antagonist

pyridoxalphosphate-6-azophenyl-2’,4’-disulfonic acid (PPADS;

10 mg/kg) or L-NAME (30 mg/kg) at a volume of 1 mL/kg.

Thereafter, they were placed in a metabolic cage with free supply of

water. During the following 24 h observation period, the rats were

kept in a light-dark-light cycle of 6, 12 and 6 h. Expelled urine was

gathered in a graded bottle below the cage and a laser Doppler

registered each drop of urine continuously. This allowed the urine

39

output, number of micturitions per hour and voided volume per

micturition to be calculated. Total water consumption was also

measured after each experiment. All data were recorded using a

MP100WSW data acquisition system and the Acquire software

(BioPac, Goleta, USA).

Cell cultivation

Two human urothelial cell lines, the UROtsa and T24 cell lines, were

cultivated, both growing as a monolayer. The UROtsa cell line is an

immortalized urothelial cell line derived from the ureter urothelial

lining (Petzoldt et al., 1995). It has been shown to have several

characteristics that closely resemble the intermediate layers of

normal human urothelium (Rossi et al., 2001). The T24 cell line is a

transitional carcinoma cell line that has been shown to be non-

tumorigenic (Flatow et al., 1987), and is frequently used as a cell

culture model to study urothelial cancer cell proliferation (Chen et

al., 2008; Teng et al., 2008; Nakanishi et al., 2009). Both cell lines

were grown at 37°C in a humidified atmosphere containing 5% CO2.

The UROtsa cell line was cultured in Dulbecco´s Modified Eagle

Medium (DMEM; Sigma-Aldrich, St.Louis, USA) while the T24 cell line

was cultured in McCoy’s 5a medium (LGC Promochem, Boras,

Sweden). Both mediums were supplemented with 10% fetal bovine

serum (Sigma-Aldrich, St.Louis, USA) and 1% penicillin/

streptomycin (PEST; PAA Laboratories, Pasching, Austria). Medium

was renewed every other day, and when the cells reached 90%

confluency they were split to a 1:3 ratio.

Measurement of proliferation

In order to measure the proliferation rate of the UROtsa and T24 cell

lines, the MTT Cell Proliferation Assay was utilized. In this assay,

confluent (90-95%) cells were trypsinized and resuspended in

growth medium at a concentration of 400 000 cells per mL. This

40

concentration was determined, by a standard procedure, to yield a

linear relationship between the number of cells and absorbance for

both cell lines. The cells were subsequently pipetted (100 µL) into

separate wells. Two hours later, drugs were added to the wells at a

volume of 10 µL, and the plate was incubated overnight at 37°C in a

5% CO2-atmosphere. Untreated cells and medium not containing

cells were used as control and blank, respectively. The following day,

MTT Reagent (10 µL), which enabled a spectrophotometrical

quantitative measurement of the proliferation rate, was added to

each well. The plate was then returned to the incubator for

approximately three hours. In order for the cells to lysate, a

detergent reagent (100 µL) was added to each well and the plate was

put in dark, at room temperature, overnight. The proliferation rate

(i.e. number of cells), equivalent to the amount of absorbance at

570 nm, was measured the following day.

Materials

Drugs

The drugs employed in this thesis were buprenorphine

hydrochloride (Temgesic®, Apoteket AB, Stockholm, Sweden),

cyclophosphamide monohydrate (CYP), collagenase type I

(Invitrogen, Paisley, UK), acetyl- -methylcholine chloride

(methacholine; MeCh), carbamylcholine chloride (carbachol),

nicotine hemisulfate salt (nicotine), 4-diphenylacetoxy-N-

methylpiperidine methobromide (4-DAMP), p-fluoro-hexahydro-sila-

diphenidol hydrochloride (pFHHSiD), pirenzepine dihydrochloride

(pirenzepine), pyridoxalphosphate-6-azophenyl-2’,4’-disulfonic acid

(PPADS), Nω-nitro-L-arginine (L-NNA), Nω-nitro-L-arginine methyl

ester hydrochloride (L-NAME), chlorbenzoyl-5-methoxy-2-

methylindole-3-acetic acid (indomethacin), phenylephrine

hydrochloride (phenylephrine), isoprenaline hydrochloride

41

(isoprenaline), phentolamine methansulphate (phentolamine),

doxazosin mesylate (doxazosin), propranolol hydrochloride

(propranolol; ICI Pharmaceuticals, London, UK), N-[[3-[(2S)-2-

Hydroxy-3-[[2-[4-[(phenylsulfonyl)amino]phenyl]ethyl]amino]-

propoxy]phenyl]methyl]-acetamide (L-748,337; Tocris, Ellisville,

USA), 3-(2-Ethylphenoxy)-1-[[(1S)-1,2,3,4-tetrahydronaphth-1-yl]-

amino]-(2S)-2-propanol oxalate salt (SR59230A), epidermal growth

factor (EGF) and heparin-binding EGF-like growth factor (HB-EGF).

Unless otherwise stated, all drugs were purchased from Sigma-

Aldrich (St Louis, USA).

Solutions and reagents

The solutions and reagents used in this thesis for cell cultivation and

cell proliferation assays were phosphate buffered saline (PBS; Sigma-

Aldrich, St Louis, USA), Dulbecco´s Modified Eagle Medium (DMEM;

Sigma-Aldrich, St.Louis, USA), McCoy’s 5a medium (LGC Promochem,

Boras, Sweden), fetal bovine serum (Sigma-Aldrich, St.Louis, USA),

penicillin/streptomycin (PEST; PAA Laboratories, Pasching, Austria),

MTT reagent (ATCC, Manassas, VA, USA), MTT detergent (ATCC,

Manassas, VA, USA) and trypsin (PAA laboratories, Pasching,

Austria).

Statistical calculations

Paper I

Comparisons were made between raw data or between relative data.

Raw data comparisons were made when increases in bladder

pressure (Δ bladder pressure) are compared between two groups.

Relative data comparisons were made when comparing the effect

that 4-DAMP had (% decrease) on basal contraction to MeCh.

42

Statistical significance was determined by Student’s t-test for paired

or unpaired data.

Paper II

Statistical comparisons using one-way analysis of variance (ANOVA),

followed by the Bonferroni correction test, was made between raw

data of mean contractile responses to carbachol in repetitive rounds.

When comparing raw data (of mean contractile responses to

carbachol) between control and CYP pre-treated bladder

preparations, Student’s t-test for unpaired data was used to

determine statistical significance. Schild plots and pA2-values were

estimated by obtaining dose ratios of full carbachol response curves

from three antagonist concentrations (Arunlakshana & Schild, 1959).

The estimated Schild slopes were compared to unity utilizing a built-

in test in the GraphPad software (GraphPad Software Inc., San Diego,

USA). Likewise, pharmacodynamic modeling was performed by

applying built-in models for nonlinear regression.

Paper III

One- or two-way ANOVA, followed by the Bonferroni correction test,

was used to determine statistical significance when comparing either

(1) data measured at several time points in the saline pre-treated

(control) group to data measured at several time points in the CYP

pre-treated group, (2) data from saline treated (control) rats to data

from rats treated with an antagonist or L-NAME, (3) data from one

treatment group to combined data from two treatment groups or (4)

data from saline pre-treated rats to data from CYP pre-treated rats

where both groups also received equivalent treatment with 4-DAMP.

Paper IV

In this paper three types of comparisons were made. Either (1) the

43

proliferation rate of UROtsa cells treated with an agonist or

antagonist was compared to the proliferation rate of T24 cells

treated with the same agonist/antagonist, (2) the proliferation rate

of cells treated with an agonist or antagonist was compared to the

proliferation rate of untreated (control) cells or (3) the proliferation

rate of cells treated with both an antagonist and an agonist was

compared to the proliferation rate of cells treated with solely the

agonist. Statistical significance was determined by Student’s t-test for

paired or unpaired data.

44

Results and Discussion

Paper I

Cholinergic nitric oxide release from the urinary bladder

mucosa in cyclophosphamide-induced cystitis of the

anaesthetized rat

In anaesthetized rats, MeCh (0.5 – 5 µg/kg i.v.) generated almost

similar bladder contractions in saline and CYP pre-treated rats with

an intact urothelium. This finding was in contrast to previous

findings in vitro, in which significant differences have been reported

(Giglio et al., 2005b; Giglio et al., 2007). However, this discrepancy

might be explained by a combination of several factors. Firstly, in

vivo the intravesical pressure can only reach a certain level before

the micturition reflex is initiated. This may have prevented the

expected differences in maximal detrusor contractility. Secondly, the

largest dose of MeCh led to a dramatic drop in blood pressure which

could have affected bladder pressure (i.e. lowered the maximal

response). Thirdly, in vivo MeCh reaches primarily the detrusor, as it

is introduced to the bladder via the blood stream, while in vitro

carbachol has equal access to detrusor and urothelium. The

inhibitory CYP-induced effects seen in vitro might therefore be

imperceptible in vivo.

When the response to MeCh (0.5 – 5 µg/kg i.v.) was tested in rats in

the presence and absence of the urothelium, larger contractions

were observed in CYP pre-treated bladders (p < 0.05; n = 5) in the

absence of the urothelium. Meanwhile, the contractions were

unaffected by removal of the urothelium in normal bladders. When

examining the dose dependent effects of MeCh on CYP pre-treated

bladders in the presence of L-NAME, no contractile alterations arose

45

by removal of the urothelium. These results indicate that there is a

relaxatory factor present in the urothelium of CYP pre-treated

bladders, and that this factor disappears upon NOS blockade.

Figure 6. Contraction of intact inflamed bladders after successively larger

doses 4-DAMP (0.1 – 1000 μg/kg). Mean contractile responses to MeCh (2 μg/kg)

in the absence () and presence () of L-NAME (30 mg/kg). Contraction is

expressed as percentage of basal response. Vertical bars represent the SEM. * p<0.05

Increasing doses of 4-DAMP (0.1 - 1000 µg/kg i.v.) could dose

dependently inhibit bladder contractions evoked by MeCh (2 µg/kg

i.v.) in both saline and CYP pre-treated bladders. This inhibition was

unaffected by the presence of L-NAME as well as the removal of the

urothelium in saline pre-treated rats. However, in intact CYP pre-

treated bladders, blockade of NOS yielded larger contractions

(Figure 6). These results showed that blockade of NOS affected

bladder contractions in cystitic but not in normal bladders. Since

NOS-blockade yielded larger contractions in cystitic bladders, the

presence of a relaxatory NO-component was indicated. However, the

source of this relaxatory NO was not revealed. For instance, it could

be present in both the urothelium, suburothelial vessels and

parasympathetic neurons. To examine this we ran experiments on

46

urothelium-denuded CYP pre-treated bladders in the presence and

absence of L-NAME (Figure 7). The increased contractions to MeCh

seen in cystitic bladders in the presence of L-NAME were abolished

by the removal of the urothelium. This showed the presence of a

relaxatory factor in the urothelium which was released upon

muscarinic receptor activation.

Figure 7. Contraction of urothelium-denuded inflamed bladders after

successively larger doses 4-DAMP (0.1 – 1000 μg/kg). Mean contractile

responses to MeCh (2 μg/kg) in the absence () and presence () of L-NAME

(30 mg/kg). Contraction is expressed as percentage of basal response. Vertical bars

represent the SEM.

Pre-treatment with CYP is a well established method for inducing

hemorrhagic cystitis (deVries & Freiha, 1990). The macroscopical

signs of successful CYP pre-treatment, i.e. bladder wall

thickening/edema and hemorrhages, were present in all CYP pre-

treated bladders. Removal of the urothelium by treatment with

collagenase could potentially damage the suburothelium, affecting

the results in an unknown fashion. However, immunohistochemical

examinations revealed that the suburothelium remained intact in all

47

bladders and that 80-90% of the urothelium was removed by the

procedure. Further, damages caused by urothelial denudation should

render a lower contractile response, which was not the case.

Detrusor overactivity (DO) and increased frequency are two of the

most common features of CYP-induced cystitis in both humans and

animals. During our experiments, we observed small spontaneous

contractions that were more frequent in cystitic bladders, especially

after treatment with L-NAME. In the state of anaesthesia, these

spontaneous contractions were too small to elicit a micturition.

However, it cannot be excluded that these contractions could yield

micturition events in the conscious rat, thereby linking the DO to

increased frequency. Many patients with DO do not respond to

antimuscarinic treatment. Minaglia et al. (2005) showed a link

between patients with DO that are refractory to antimuscarinic drugs

and concomitant IC. This non-responsiveness to antimuscarinic

treatment could perhaps in part be explained by our findings.

The current investigation shows that CYP pre-treatment alters the

cholinergic response of the urinary bladder. We show that NO is

present in the urothelium during CYP-induced cystitis and that this

NO exerts an inhibitory effect on detrusor contractility. Further, we

suggest that NO is released upon cholinergic stimulation, possibly

through activation of M5 and/or M3 receptors.

48

Paper II

The muscarinic M5 receptor is the primary mediator of

urothelium-derived nitric oxide effects in the rat urinary

bladder

In bladder strips from CYP pre-treated rats, the maximal contractile

response to carbachol (0.01 - 100 µM) was significantly reduced in

comparison to normal bladder strips (Figure 8; 19.3 ± 1.0 mN and

14.3 ± 1.2 mN in saline and CYP pre-treated strips, respectively;

p < 0.01).

Figure 8. Contraction of intact urinary bladders. Mean contractile responses to

carbachol (0.01 - 100 µM) of isolated detrusor strip preparations from saline (●) and

CYP (■) pre-treated rats. Vertical bars represent the SEM. * p<0.05; ** p<0.01

A comparison of the EC50-values also showed a significant right-shift

of the concentration-response curve in CYP pre-treated bladder

strips (logEC50 = -6.1 and -5.4 in saline and CYP pre-treated rats,

respectively; p < 0.001). The right shift, as well as the difference in

maximal contractile response, was abolished by removal of the

urothelium (Figure 9). Administration of the NOS inhibitor L-NNA

49

also normalized the contractile differences between normal and

cystitic bladder strips (Figure 10). The same normalization of

contractile responses to carbachol appeared when comparing

normal and cystitic urothelium-denuded bladder strips in the

presence of L-NNA.

Figure 9. Contraction of urothelium-denuded urinary bladders. Mean

contractile responses to carbachol (0.01 - 100 µM) of isolated detrusor strip

preparations from saline (●) and CYP (■) pre-treated rats. Vertical bars represent

the SEM.

These findings further strengthen the idea that in CYP-induced

cystitis the urothelium exerts a relaxatory influence on the detrusor.

Further, since it was possible to block this influence by

administration of L-NNA, NO is likely to be the primary inhibitory

factor. Other possible relaxatory factors include prostaglandins and

adenosine. Particular interest should be taken to the latter, as it has

been shown to exert a relaxatory influence on the detrusor

(Aronsson et al., 2010). However, we could not see any effect of

indomethacin, an inhibitor of COX, on the contractile responses, and

studies have shown that the relaxatory effect of adenosine decreases

in cystitis (Vesela et al., 2010).

50

Figure 10. Contraction of intact urinary bladders in the presence of L-NNA

(100 µM). Mean contractile responses to carbachol (0.01 - 100 µM) of isolated

detrusor strip preparations from saline (●) and CYP (■) pre-treated rats. Vertical

bars represent the SEM.

The carbachol induced contractions were concentration-dependently

inhibited by both 4-DAMP, the muscarinic M3 antagonist pFHHSiD

and the muscarinic M1 antagonist pirenzepine. While pFHHSiD and

pirenzepine inhibited the contractile responses of saline and CYP

pre-treated rats in a similar fashion, 4-DAMP inhibited the

contractile responses of CYP pre-treated bladder strips less potently

than saline pre-treated (pA2 value = 9.9 and 9.5 in saline and CYP

pre-treated bladders, respectively; p < 0.01). In the CYP pre-treated

bladders, the lowest concentration of 4-DAMP (10-10 M) even tended

to generate a left-shift of the contraction curve. By either removing

the urothelium or running the experiments in the presence of L-NNA,

the difference in inhibitory potency of 4-DAMP in CYP pre-treated

bladders was abolished. Analysis of the Schild slopes for all three

antagonists revealed that they were all significantly different from

unity in CYP pre-treated bladder strips, but not in the normal

preparations. This is in favor of the notion that a change in

contractility occurs during cystitis. Removal of the urothelium

eliminated this difference, indicating that the contractility is altered

due to changes mediated via the urothelium. Similarly, the difference

51

from unity of the Schild slopes was abolished by the presence of

L-NNA.

The antagonistic profile is different for the three examined

antagonists. While 4-DAMP inhibits M1, M3 and M5 in a similar

fashion, pFHHSiD and pirenzepine are considered mainly a M3- and

M1-antagonist, respectively (Eglen & Nahorski, 2000). The

antagonistic potency of 4-DAMP is reduced in cystitic bladder strips,

indicating that either the M1, M3 or M5 receptor (or more than one

of them) is involved in the altered contractile responses seen during

cystitis. Since neither the potency of pFHHSiD nor pirenzepine is

changed during cystitis, M5 seems to be the primary receptor

involved in this alteration. Taken together, our findings indicate that

changes occur during CYP-induced cystitis that alter NO-mediated

effects via urothelial M5 receptors.

Correlation plots for the pA2-values obtained in the study versus

–Ki values reported in the literature for the five muscarinic receptors

support the generally accepted idea that detrusor contraction is

mediated mainly by stimulation of muscarinic M3 receptors, both in

normal and inflamed bladders (r2 for M3 = 0.98 and 0.93 in normal

and inflamed bladder strips, respectively). Thus, as expected, these

plots do not support a notion that M5 receptors are directly involved

in the contractile response of the detrusor. However, the correlation

plots show that during CYP-induced cystitis, the M3-mediated

contraction seems to be less dominant, indicating the influence of

some other receptor or factor on contraction.

It is important to keep in mind that there may be differences

between CYP-induced and naturally occurring cystitis, as well as

inter-species disparities. Studies on bladder strips from cats have

shown that during naturally occurring cystitis, activation of

urothelial muscarinic receptors may actually lead to increased

contractions at low agonist doses (Ikeda et al., 2009). Therefore,

52

extrapolation of the interpretation of the data in this study to man

must be performed with caution. Further, one also has to keep in

mind that these are studies performed in an organ bath setup,

thereby excluding the possibility for compensatory effects via the

nervous system or changes in blood pressure.

To summarize, this study shows that the contractile changes seen in

CYP-induced cystitis are mediated via stimulation of muscarinic M5

receptors and the following release of NO. Further, this study agrees

with several previous studies that conclude that detrusor contraction

in the healthy rat bladder is mediated almost entirely via muscarinic

M3 receptors.

53

Paper III

Pharmacological modulation of the micturition pattern in

normal and cyclophosphamide-pretreated conscious rats

The micturition pattern of conscious rats was assessed during 24 h,

while the rat was placed in a metabolism cage. In the saline treated

rats, pre-treatment with CYP (100 mg/kg) significantly reduced the

volume per micturition over 24 h (Figure 11; 0.57±0.19 vs. 0.85±0.11

mL in CYP and saline pre-treated rats, respectively; p < 0.01; n = 6

and 10, respectively). At the same time, the water intake was not

significantly different between these groups, or between any other

groups.

Figure 11. Volume of urine produced at each micturition event in saline

(●; n=10) and CYP (■; n=6) pre-treated rats. Values are given as means ± SEM.

Points indicate time periods 0 – 6 h, 6 – 18 h and 18 – 24 h. ** p<0.01 (overall

difference between the groups (ANOVA))

Thus, changes occurred during CYP-induced cystitis that reduced the

micturition volume per event. This could depend on alterations in

54

the contractile threshold of the detrusor, or solely be a result of a

decreased detrusor contractile ability. The latter would increase the

amount of residual urine, which has been shown to occur during

CYP-induced cystitis (Ito et al., 2007). We could also see a slight, but

not significant, increase in micturition frequency in CYP pre-treated

rats. Since the volume per micturition is lower in CYP pre-treated

rats, at the same time as the water intake is unchanged, and other

studies have shown an increase in frequency during CYP-induced

cystitis (Smaldone et al., 2009), we would have expected a significant

increase in micturition frequency. However, in many other studies

observations have been made over shorter time periods (< 12 h). In

our study, measurements were made over a 24 h period, which may

conceal temporary changes occurring during shorter periods.

Others have shown that CYP-induced cystitis can cause bladder

overactivity (Borvendeg et al., 2003). During CYP-induced cystitis, an

increase in both the release of urothelial ATP and of the expression

of eNOS and muscarinic receptors has been shown to occur (Giglio et

al., 2005b; Smith et al., 2005). In the current study, in CYP pre-

treated rats, treatment with L-NAME (30 mg/kg) led to an increase in

micturition frequency (Figure 12a; 2.53 ± 0.36 vs. 1.25 ± 0.17

events/h in L-NAME and saline treated rats, respectively; p < 0.05;

n = 6 in both groups). In saline pre-treated rats, L-NAME did not

affect any micturition parameter. This is in line with findings in other

studies that show that intravesically administered NO-donors can

abolish the increased frequency seen in CYP pre-treated rats (Ozawa

et al., 1999). Our results, and those of others, show that in CYP-

induced cystitis, NO exerts an inhibitory (relaxatory) effect on the

detrusor.

55

Figure 12. Effects of treatments on micturition parameters in cystitis.

Frequency of micturition events (a) and volume of urine produced at each

micturition event (b) of CYP pre-treated rats during the 6 – 18 h dark period. Forty-

eight hours after CYP pre-treatment, saline (1 mL/kg i.p.; n=10), 4-DAMP (1 mg/kg

i.p.; n=6), L-NAME (30 mg/kg i.p.; n=6) or PPADS (10 mg/kg i.p.; n=6) was

administered. Values are given as means ± SEM. * p<0.05

The muscarinic antagonist 4-DAMP affected micturition parameters

of CYP but not saline pre-treated rats. The volume per micturition of

CYP pre-treated rats during the 6 – 18 h dark period was

significantly increased by treatment with 4-DAMP (Figure 12b;

1 mg/kg; 0.75 ± 0.05 and 0.51 ± 0.09 mL in 4-DAMP and saline

treated rats, respectively; p < 0.05; n = 6 and 10, respectively).

Similarly, in paired comparisons, treatment with different doses of

4-DAMP (10 - 1000 µg/kg) eliminated the significant difference in

volume per micturition between saline and CYP pre-treated rats.

Even though non-significant, the frequency of CYP pre-treated rats

was increased, rather than decreased, by treatment with 4-DAMP

(Figure 12a). This finding is in line with studies in man that report

that patients with DO are non-responsive to antimuscarinic

treatment (Minaglia et al., 2005). It is evident that treatment with

56

antimuscarinic drugs has limited effects in the healthy bladder of

rats, which is in line with previous studies (Modiri et al., 2002).

However, in the cystitic bladder, 4-DAMP increases the volume per

micturition, perhaps via an increase of detrusor contractility. The

absence of effect of an antimuscarinic drug in the normal rat bladder

is likely, at least partly, due to compensatory effects from the

purinergic system.

Treatment with a combination of 4-DAMP (1 mg/kg) and L-NAME

(30 mg/kg) did not affect any of the micturition parameters

differently than separate administration of the two drugs, in neither

saline nor CYP pre-treated rats. However, when combining 4-DAMP

(1 mg/kg) with PPADS (10mg/kg), a synergistic effect was seen on

the voiding frequency in CYP pre-treated rats during the 6 – 18 h

dark period. This could indicate that at least part of the effects of

muscarinic antagonists and those of NOS blockers are mediated via

the same pathway, while the effects of purinergic antagonists are

mediated via a separate pathway. The reason for why a synergistic

effect is only seen during the dark period is likely due to the fact that

it is during this period that the rats are awake, and thus the

micturition activity is the highest and most likely to reveal

differences in micturition parameters.

When given separately to CYP pre-treated rats, treatment with

4-DAMP leads to increased volumes per micturition, while treatment

with L-NAME leads to increased micturition frequency. Even though

these two parameters are not totally interlinked, they indicate an

opposite effect of 4-DAMP and L-NAME in CYP pre-treated rats. To

understand the discrepancies between a combinatory treatment and

that of 4-DAMP and L-NAME alone, one has to remember that

4-DAMP may exert its effects via two separate pathways; (a) via

blockade of muscarinic receptors in the efferent and/or afferent

pathways, leading to decreased detrusor activity, and (b) via

blockade of urothelial muscarinic receptors, which according to our

57

studies in vivo and in vitro leads to a decreased relaxatory influence

of NO on the detrusor. This kind of dual effect of 4-DAMP would

explain our findings.

Experiments on wake, freely moving rats present certain problems

when examining drugs that have a relatively small impact on

micturition parameters. More specifically, the interindividual

variations among rats are sometimes larger than the effects of the

studied drugs, making it impossible to statistically detect minute

changes. However, this observable fact strengthens the correctness

in those significant differences one does find.

The current study indicates that changes occur during CYP-induced

cystitis that not only affects the micturition parameters, but also the

effects of antimuscarinic, antipurinergic and antinitrergic drugs. It is

possible to reverse the altered micturition parameters in the

inflamed rat bladder by treatment with an antimuscarinic drug,

indicating the strong involvement of these receptors in the

alterations occurring during cystitis. Further, this study shows that

there is a connection between the effect of antimuscarinic and

antinitrergic drugs. Thus, this study strengthens the previously

proposed idea of an increased importance of NO, which is released

upon muscarinic receptor stimulation, during CYP-induced cystitis.

58

Paper IV

Characterization of receptor mediated proliferation in the

human bladder urothelial UROtsa and T24 cell lines

Studies on various types of gastrointestinal inflammation have

shown that chronic inflammation of the bowel together with

increased proliferation of the mucosa is strongly associated with the

development of cancer (Ament, 1975; MacDonald, 1992). Treatment

with CYP causes similar proliferative changes in the urothelium

(Zupancic et al., 2009). Further, more than 90% of all urinary bladder

cancer originates from the urothelium. When studying this disease,

others have reported a link between chronic inflammation and

urothelial transformation (Talar-Williams et al., 1996; Michaud,

2007). It has also been shown that the expression and/or sensitivity

of certain receptors in the urothelium can be altered in the state of

inflammation and transformation (Giglio et al., 2005b; MacLaine et

al., 2008).

The proliferative effects of adrenergic, muscarinic, nicotinic and EGF

receptor agonists were examined in an immortalized normal human

bladder urothelial cell line, designated UROtsa, and a malignant

human bladder urothelial cell line, designated T24. Regarding the cell

culturing, all experiments were performed during passage 4 – 10.

This should be well within the time frame where one would expect

cell properties to stay consistent.

The non-selective β-adrenoceptor agonist isoprenaline

concentration-dependently increased the proliferation rate of both

the UROtsa and T24 cell line (Figure 13a; pEC50 = 3.0 and 3.1,

respectively). This proliferative effect could not be antagonized by

the β1/β2-selective antagonist propranolol (2 x 10-5 M) in neither the

UROtsa nor T24 cell line. However, the selective β3-adrenoceptor

59

antagonist L-748,337 (1 x 10-6 – 5 x 10-5 M) showed a concentration-

dependent inhibition of the proliferative effect of isoprenaline in

both the UROtsa and T24 cell line (at 2 x 10-5 M p < 0.01 and 0.05 in

the UROtsa and T24 cell line, respectively; n = 6 in both groups).

SR59230A (2 x 10-5 M) had no effect in the UROtsa cell line, but

significantly decreased the proliferative response to isoprenaline in

the T24 cell line (p < 0.05; n = 6). SR59230A was first believed to be a

β3-selective antagonist, but later studies have shown that it also can

function as both an α1-adrenoceptor antagonist and an unselective β-

adrenoceptor antagonist (Hoffmann et al., 2004; Bexis & Docherty,

2009). Further, when tested separately, SR59230A significantly

decreased the basal proliferation rate of T24, but not UROtsa, cells

(p < 0.0001; n = 6). Therefore, the inhibitory effect of SR59230A on

the T24 cell line is likely to be the result of SR59230A acting as a

dirty drug.

Figure 13. Proliferative response to (a) isoprenaline and (b) phenylephrine of the

UROtsa ( ; n=10) and T24 (; n=7) cell lines. Vertical bars represent the SEM.

* p<0.05; ** p<0.01; *** p<0.001

60

These results indicate that the increased proliferative response to

isoprenaline in the UROtsa and T24 cell lines is mediated mainly via

the β3-adrenoceptor. Interestingly, β3-agonists, which have been

suggested as treatment for OAB, have been shown to induce cystitis

in rats (Waghe et al., 1999). Based on our finding in urothelial cells,

we would expect such treatment to yield thickening of the bladder

mucosa, one of the characteristic symptoms of cystitis. The reason

for why it was not possible to block the entire proliferative effect of

isoprenaline with L-748,337 could depend on one of two things (or

both of these). Either the effect is partly mediated via other pathways

than activation of the β3-adrenoceptor, or the concentrations of

L-748,337 chosen for the experiments (1 x 10-6 – 5 x 10-5 M) were not

high enough to block the entire proliferative effect. The range of

concentrations of L-748,337, and that of all other agonists and

antagonists, was based on pilot experiments, and doses showing

signs of toxic effects were excluded. Unfortunately, it was therefore

not possible to test higher concentrations of L-748,337.

In the presence of the α1-adrenoceptor agonist phenylephrine, a

decrease of the proliferation rate was seen in both the UROtsa and

T24 cell line (Figure 13b; pIC50 = 2.2 and 1.7, respectively; n = 6). At

the highest concentration (1 x 10-2 M), there was a significantly

greater anti-proliferative effect in the UROtsa cell line (Figure 13b;

p < 0.0001; n = 6). When it comes to anti-proliferative effects, one

cannot exclude that the response is not simply a toxic effect. This is

further highlighted by the fact that neither the unselective α-

adrenergic antagonist phentolamine (10-9 – 10-4 M) nor the α1-

selective adrenoceptor antagonist doxazosin (10-9 – 10-4 M) could

block the effect of phenylephrine (at 5 x 10-3 M). However, if the anti-

proliferative effect of phenylephrine was not mediated via receptor

activation, the impact should be equal in the two cell lines. Further,

the concentrations of α-adrenergic antagonists used in this study

could have been too low to inhibit the effect of phenylephrine at

61

5 x 10-3 M. Unfortunately, it was not possible to use higher

concentrations of antagonists due to toxic effects.

The expression of muscarinic receptors seems to be different in the

two studied cell lines. While all subtypes (M1 - M5) have been shown

to be present in the UROtsa cell line (Tyagi et al., 2006), another

study could only detect mRNA for the M2 and M5 receptors in the

T24 cell line (Tully et al., 2009). As we have shown in a previous

study on rats, the expression of muscarinic receptors, in particular

the M5 subtype, was altered in the state of CYP-induced cystitis

(Giglio et al., 2005b). Thus, one of the possible links between

inflammation and bladder cancer could be alterations in the

expression of muscarinic receptors. We could, however, not see any

functional effect of this altered expression, as the non-selective

muscarinic agonist methacholine did not affect the proliferation rate

of neither the UROtsa nor T24 cell line. One plausible explanation for

the lack of proliferative effects upon activation of muscarinic

receptors could be due to two or more effects cancelling each other

out. I.e., by employing a non-selective agonist, activation of one

subtype might increase proliferation, while activation of another

subtype might decrease the rate of cell division. Future experiments

should be designed to elucidate this possibility.

Nicotine, and the activation of the nicotinic receptor, has been

associated with the development of bladder cancer and modulation

of the proliferation of malignant urothelial cells (Chen et al., 2008).

However, we could not see any effect of nicotine (1 x 10-9 –

1 x 10-7 M) in neither the UROtsa nor T24 cell line. This could be due

to toxicity problems, which only rendered the possibility to use very

low concentrations.

Activation of the EGF receptor by EGF or HB-EGF is closely linked to

increased cell proliferation, and their involvement in the

transformation of urothelial cells has been suggested (Adam et al.,

62

2003). Several other studies have shown an increased proliferation

rate upon EGF receptor activation, but despite using wide ranges of

concentrations for both EGF (5 – 500 ng/mL) and HB-EGF

(1 – 200 ng/mL), we could not see this effect in neither the UROtsa

nor T24 cell line. This could possibly be explained by studies that

have shown that only the autocrine release, and not the exocrine

addition, of EGF or HB-EGF can affect the proliferation rate of

urothelial cells (Southgate et al., 1994; Rebel et al., 1995; Freeman et

al., 1997; Sterle et al., 1997). Further, other studies have suggested

that activation of the EGF receptor only increases the proliferation

rate of urothelial cells during challenges such as wound repair

(Varley et al., 2005).

Studies on cell cultures are beneficial in the sense that they offer an

experimental setup that allows the study of one specific target in one

specific cell type. However, one must always draw conclusions from

cell culture studies with caution. The proliferation of urothelial cells

is most likely an interplay between not only the urothelial cells

themselves, but also other cell types. Findings in cell cultures should

therefore, when possible, be verified in vivo.

The results in this report show the importance of adrenergic

receptors in the regulation of urothelial cell proliferation, both in a

normal and a malignant cell line. Further, since the UROtsa cell line is

affected to a greater extent than the T24 cell line by both α- and β-

adrenoceptor stimulation, we show that the impact of adrenergic

receptor stimulation on cell proliferation is decreased in the

malignant state. This report also examines the impact of muscarinic,

nicotinic and EGF receptor stimulation, but the current results do not

support the involvement of any of these in urothelial cell

proliferation during unchallenged conditions.

63

Concluding remarks

A primary aim of this thesis was to elucidate the cause of the

difference in contractility between the normal and CYP-inflamed

urinary bladder. By piecing together several indirect and direct

findings, we show that during CYP-induced cystitis there is an

increased influence of NO on bladder contractility. Further, our

accumulated findings point to this NO being released upon activation

of muscarinic M5 receptors in the urothelium.

Figure 14. Influence of the urothelium on the healthy (left) and inflamed

(right) urinary bladder. In both, contraction of the detrusor is mediated mainly by

activation of muscarinic M3 receptors. During CYP-induced cystitis, a significant

amount of nitric oxide (NO) is released from the urothelium upon activation of

muscarinic M5 receptors, thereby dampening the contractile properties of the

detrusor. In the inflamed bladder, NO has also been suggested to act on and

modulate the activity of afferent nerve fibers in the suburothelium. Apart from NO,

ATP is released from the urothelium upon stretch and/or muscarinic receptor

activation, affecting afferent nerve fibers in the suburothelium and detrusor. The

impact of ATP in man has been suggested to be greater in the state of cystitis.

M - muscarinic receptor; M3 - muscarinic receptor of the M3 subtype; M5 -

muscarinic receptor of the M5 subtype; NO - nitric oxide; ACh - acetylcholine.

64

Much of the research on the urothelium has focused on its role as an

integrator of afferent signaling. This led to that early studies on the

involvement of the urothelium during cystitis mainly examined the

effects that factors released from the urothelium had on afferent

nerves. However, the results presented in this thesis show that

urothelial factors have impact on not only afferent, but also efferent

mechanisms. It is also currently shown that the significance of this

impact can be altered during disease states such as cystitis. This

knowledge might highlight numerous new targets for future

pharmacological treatments. For instance, there is a debate whether

up-regulation of NO during cystitis is solely a result of the

inflammation, or if it is actually part of the cause of the inflammation

(Souza-Fiho et al., 1997). This is a matter of great interest,

particularly regarding the development of new pharmacological

therapies for IC, which are needed. A major problem today is the

uncertainty in the diagnosis of IC. Several factors have been

suggested as early markers for IC, but so far no diagnostic tests have

been developed. If an early marker for IC was found, and NO plays an

important part in the development of inflammation, it might be

possible to prevent IC by inhibiting the increased impact of NO

during cystitis. Future experiments should be conducted in order to

elucidate the development of inflammation, and if it is possible to

influence this development.

A key concern for our studies was the proper induction of cystitis. In

order for our data to be translatable, it was relevant for us that the

cystitis we studied was similar to IC as it appears in man. Several

animal models have been established to study experimental cystitis,

of which induction with CYP is the most common (deVries & Freiha,

1990). There are many similarities between CYP-induced cystitis and

IC. For one, CYP-induced cystitis gives symptoms that are very

similar to IC. Secondly, none of them give rise to inflammation due to

infection. Thirdly, CYP-induced cystitis, just as IC, has been reported

to sensitize afferent pathways (Yu & de Groat, 2008). Further, IC is

65

associated with an altered release of NO and ATP. During CYP-

induced cystitis, an up-regulation of both endothelial nitric oxide

synthase (eNOS) and muscarinic receptors, mainly of the M5

subtype, has been shown (Giglio et al., 2005b). Also, both altered

release of ATP and increased frequency have been shown to arise

during CYP-induced cystitis (Wood et al., 2001; Smith et al., 2005).

However, there are also certain differences between IC and CYP-

induced cystitis. For instance, ultrastructural studies of bladder

specimens from IC patients has shown a disrupted barrier function of

the urothelium (Elbadawi & Light, 1996). This is not equally

apparent in CYP-induced cystitis, where the common observation is a

thickening of the bladder mucosa. There may also be differences in

the initiating causes of inflammation. In CYP-induced cystitis the

inflammatory response is evoked by the presence of chemical

irritants in the urine, while the exact cause for IC remains unclear.

The results presented in this thesis may also be applicable to other

bladder disturbances than IC. In man, the classic symptom of DO is

frequency. Treatment for patients suffering from frequency is today

focused on antimuscarinic drugs. However, several studies have

shown limited long-term clinical effects of these drugs (Andersson,

2009). A possible goal for the future treatment of symptoms of

frequency could be via regulation of NO release. If our data are

translatable to man, a compound selective for the muscarinic M5

receptor might improve quality of life for millions of patients

suffering from inflammation-induced DO.

As mentioned previously, a recurrent observation during our studies

was an increased proliferation of the urothelium upon CYP-

treatment. Concomitantly, an increase of the expression of

muscarinic M5 receptors occurs in the urothelium (Giglio et al.,

2005b). This same link, between increased proliferation and

expression of M5 receptors, is observed in other diseases such as

Sjögrens syndrome (Ryberg et al., 2008). Despite these studies

66

showing a potential link between proliferation and increased

expression of M5 receptors we could not show any effect of a

muscarinic agonist on urothelial cell proliferation. The expression of

all five muscarinic receptors has been shown in normal human

urothelium (Table 1). However, in order to validate these findings,

future studies should be performed to elucidate receptor expression

in the UROtsa and T24 urothelial cell lines. Furthermore, future

proliferation experiments should be performed in order to

investigate if other results could have been obtained by the use of

selective agonists and antagonists for these receptors.

67

Acknowledgements

My sincere gratitude goes out to all of you whom have helped me

complete this thesis. You know who you are. However, some of you I

would like to thank in particular.

First and foremost I would like to thank my supervisor, Gunnar

Tobin. Without your help and encouragement I would have never

even begun my PhD-studies, let alone completed them. I owe you

one.

My co-supervisor, Daniel Giglio. Thank you for the many pointers and

good scientific discussions.

My bladder friend, Patrik Aronsson. Thank you for your company,

friendship and all our great discussions. And thank you for putting up

with me during our trips.

My fellow PhD-student in our group, Anders Ryberg. Thank you for

teaching me immunological methods in the lab and that comic strips

can be considered fine art.

Group members Hanna Asklund, Martin Johnsson and Christine

Ahlström. Thank you for fun lunches and help with my papers.

Co-authors Anna Wilhelmson, Davoud Eskandari, Deya Doufish and

Anette Lampert. Thank you for your help in gathering data for my

papers and for you good company.

My pharmafriends from the Czech Republic, Renata Vesela, Ondrej

Soukup and Pavel Jeřábek. Thank you for helping me with my papers.

And thank you for teaching me that dumplings are an essential part

of life.

Professor Scott Garrett, Univeristy of South Dakota, for kindly

providing me with the UROtsa cell line without any request for

compensation.

68

To Hans Nissbrandt and Christer Nilsson at the department of

pharmacology. Thank you for discussing sports, my favorite interest,

with me. I need that sometimes.

To all other co-workers at the department of pharmacology. Thank

you for proving such a stimulating and positive surrounding.

To my friends at other parts of the University, Johan Bourghardt,

Louise Strandberg and Marie Kalm. Thank you for numerous fun

lunches

To former and present members of the PhD committee, in particular

Malin von Otter and Karin Hultman. Thank you for your

commitment.

To my parents, for guiding me to where I am today. Thank you for

your endless support.

To my son, William. You bring a smile to my face each and every day.

Thank you for teaching me that one should not take the voluntary

control of the bladder for granted.

Finally, but mostly, to my beloved Elin. Thank you for always being

there, for always listening to me (even when I ramble) and for

showing me what is most important in life. I love you with all my

heart.

This thesis was supported by grants from Anders Otto Swärd

Foundation/Ulrika Eklund Foundation, Ferrings och Svenska Enures

Akademin, Lars Hierta Foundation, Wilhelm och Martina Lundgrens

Vetenskapsfond, Karl och Annie Leons Minnesfond and the Magn.

Bergvall Foundation

69

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